The pathophysiology of diabetic neuropathy is poorly understood. We reported previously that sera from patients with type 2 diabetes mellitus with neuropathy induced programmed cell death (PCD) in cultured human neuroblastoma cells via an autoantibody-mediated pathway involving activation of caspase- dependent apoptosis. Recent studies support that a caspase-independent pathway can also contribute to PCD. This process may involve activation of autophagy, a pathway that sequesters proteins and organelles in autophagosomes in response to cellular stress. We propose the novel hypothesis that autoantibodies present in the sera of type 2 diabetic patients with neuropathy sequentially activate autophagy, caspase- dependent and -independent PCD in neurons via a Fas-dependent pathway. Fas (CD95) is a member of the cell membrane-bound death receptor family. Our preliminary data support the hypothesis that agonist autoantibodies bind and activate the Fas receptor. We will monitor the development of autoantibody(-ies) and correlate their presence with established markers of autonomic and peripheral neuropathy in an early and later cohort of patients with type 2 diabetes mellitus. We will dissect the pathway(s) that induce autophagy and PCD. We propose that autophagy is an early cytoprotective response to remove injured mitochondria that progresses sequentially to caspase-dependent and -independent PCD with decrease in ATP levels. We hypothesize that autoantibody-induction of autophagy will involve incorporation of LC3n, a specific marker for autophagosomes, and activation of PI-3 kinase (class III). Cultured SH-SY5Y (human neuroblastoma cells) will be exposed to complement-inactivated sera obtained from: 1. type 2 diabetic patients with documented diabetic neuropathy, 2. Age- and gender- matched type 2 diabetic patients without evidence of neuropathy and 3. Healthy, age- and gender-matched controls. Parallel animal studies will be performed examining whether autophagy is activated in situ in primary neurons (nodose ganglia, dorsal root ganglia and myenteric plexus) obtained from the female Zucker Diabetic Fatty rat, an inducible model of type 2 diabetes and the streptozotocin-induced diabetic rat, a well validated model of diabetic neuropathy. We will also assess whether sera from diabetic rats with neuropathy induce autophagy and caspase-dependent and/or -independent PCD in cultured rat nodose ganglia, DRG and myenteric neurons compared to sera from diabetic rats without neuropathy and lean, non-diabetic controls, and examine the time-course for these events. We hypothesize that mitochondrial dysfunction and decreased levels of ATP will play a pivotal role in sequential activation of caspase-dependent and -independent PCD. Relevance to public health: These studies will lead to novel insights regarding the mechanistic basis of autoimmunity, autophagy and programmed cell death in the pathophysiology of diabetic neuropathy. .